Characterization of genes encoding diterpene cyclases responsible for phytoalexin biosynthesis in rice.

从水稻中分离出14个二萜类植物保卫素，分别为木犀草酮、植物卡介烷和谷氮素。我们已经分离到编码负责这些植物保卫素生物合成的二萜环酶的cDNA：OsCPS2、OsCPS4、OsKSL4、OsKSL7、OsKSL8、OsKSL10。这个项目是为了表征环化酶基因。1)基因表达谱：OsCPS2、OsCPS4、OsKSL4、OsKSL7、OsKSL8在根中的表达水平高于地上部。水稻果实中OsCPS2、OsCPS4、OsKSL4和OsKSL7在抽穗至开花期的表达水平显著升高。利用绿色荧光蛋白进行的瞬时检测表明，这些二萜环化酶是运输的细质体。2)同源基因的分离：从水稻KS类同源基因中分离到编码ent-pimara-8，15-二烯合成酶和ent-kaur-15-ene合成酶的OsKSL5和OsKSL6。从小麦和禾本科其他物种中分离到了OsCPS2和OsCPS4的两个同源基因。3)水稻二萜类植物保卫素与病原性的关系：稻瘟病菌侵染水稻后，6个基因的表达水平均升高。我们没有通过RNAi筛选Tos 17插入-敲除突变体和产生敲除突变体。4)比较OsCPS2和负责赤霉素生物合成的OsCPS1：异位表达OsCPS2挽救了缺失OsCPS1的突变体的严重矮化表型。这一结果表明，OsCPS1的表达调控不同于OsCPS2的表达调控。OsCPS2和OsCPS1的基本酶学性质基本相同，如二价阳离子需要量、最适pH、最适温度和动力学参数。然而，底物香叶基香叶基二磷酸和赤霉素生物合成抑制剂AMO 1618对OsCPS2的抑制作用比OsCPS1弱。

From rice 14 diterpenoid phytoalexins were isolated; momilactones, phytocassanes, and oryzalexins. We have isolated cDNAs encoding diterpene cyclases responsible for biosynthesis of these phytoalexins; OsCPS2, OsCPS4, OsKSL4, OsKSL7, OsKSL8, OsKSL10. This project was for characterization of the cyclase genes. 1) Gene expression patterns: The expression levels in roots of OsCPS2, OsCPS4, OsKSL4 OsKSL7, and OsKSL8, were higher than those in shoots. The expression levels of OsCPS2, OsCPS4, OsKSL4, and OsKSL7 were drastically increased in the rice fruits from heading stage to flowering stage. Transient assay using green fluorescence protein suggested that these diterpene cyclases are transported thin plastids. 2) Isolation of their homologues: From rice KS like homologue cDNAs, OsKSL5 and OsKSL6, which encode ent-pimara-8,15-diene synthase and ent-kaur-15-ene synthase respectively, were isolated. Two homologue cDNAs of OsCPS2 and OsCPS4 were isolated from wheat, other species in Poaceae. 3) Relationship of rice diterpene phytoalexins with pathogenic tolerance: The expression levels of six genes increased after infection by Magnaporthe grisea. We did not succeed in screening Tos 17-insertion-knock-out mutants and production of knock-down mutants by RNAi. 4) Comparison of OsCPS2 to OsCPS1 that is responsible for gibberellin biosynthesis: The severe dwarf phenotype of mutants lacking in OsCPS1 were rescued by ectopic expression of OsCPS2. This result indicated that regulation of OsCPS1 expression is different from that of OsCPS2 expression. The basic enzymatic properties of OsCPS2 were almost same as those of OsCPS1, such as divalent cation requirement, optimal pH, optimal temperature and kinetic parameter. However, the activity of OsCPS2 was suppressed by both of substrate geranylgeranyl diphosphate and a gibberellin-biosynthesis-inhibitor AMO 1618 less than that of OsCPS1.